The present invention relates generally to methods of treating viral infection, and particularly to methods for preparing specific peptide inhibitors of virus-specified proteases.
Proteases are enzymes which cleave proteins at specific peptide bonds. In living systems, highly specific proteases and complementary protease inhibitors mediate or control a broad spectrum of biological functions. For example, proteases cleave precursors to form active proteins in post-translational processing of polypeptides, provide mechanisms for zymogen activation cascade reactions such as blood coagulation, fibrinolysis, and complement reactions of the immune system, and mediate transport of selected proteins across biological membranes. Accordingly, proteases represent potential targets for therapeutic agents designed to function as specific inhibitors of protease activity.
Proteases encoded by viral genomes play a critical role in replication of many viruses. Viral proteases cleave large precursor polypeptides, produced by infected cells, into smaller protein components, or subunits, which are subsequently assembled to form functional virus structures. Lozitskii et al., Usp. Sovrem. Biol. 93:352-362 (1982), discuss the role of proteolysis in replication of avian and mammalian viruses, and have surveyed part of the literature relating to viral protease inhibitors.
Post-translational processing of viral polypeptides by a virus-specified protease occurs in replication of several important families of animal viruses, including the following:
______________________________________ Virus Family Representative Genera ______________________________________ Picornaviruses poliovirus rhinovirus coxsackievirus foot-and-mouth disease virus hepatitis A virus cardiovirus Togaviruses rubella virus yellow fever virus dengue virus equine encephalitis virus RNA tumor viruses retroviruses oncoviruses Adenoviruses various types ______________________________________
A role for a virus-specified protease has also been proposed in replication of other virus families, notably myxoviruses, paramyxoviruses, vaccinia viruses, comoviruses, and reoviruses. Korant, "Inhibition of Viral Protein Cleavage", in Antiviral Chemotherapy, Gauri, ed., (Academic Press, New York, 1981) and Korant, "Regulation of Animal Virus Replication by Protein Cleavage", in Proteases and Biological Control, (Cold Spring Harbor Laboratory, 1975) are reviews of literature relating to virus-specified proteases.
Picornaviruses, which are important pathogens in man and animals, are exemplary of viruses which encode a specific protease involved in viral reproduction.
Picornaviruses are small, non-enveloped, RNA-containing viruses which are important pathogens in man and other mammals. Prototypical of the picornavirus group are polioviruses, which are the causative agents of poliomyelitis, a well-known and devastating disease of the central nervous system. In previous decades, poliovirus epidemics caused paralytic disease in thousands of children and young adults, spurring research which led to effective immunization and near-eradication of the disease in industrialized Western nations. Today, in densely-populated regions where sanitation is primitive, poliovirus remains widespread. Although some children are affected, the majority of the population in such areas have antibodies to the major poliovirus types. In Western countries, however, the virus is much less prevalent. Occasionally, clinically significant cases arise among non-immunized individuals.
Other picornaviruses affecting man include coxsackie viruses, which have been associated with mild intestinal infections; rhinoviruses, which are associated with colds and minor respiratory infections; hepatitis A virus; and cardioviruses, implicated in encephalomyocarditis.
Foot-and-mouth disease viruses (FMDV) are a genus of picornaviruses which afflict cattle and other cloven-hooved animals. Foot-and-mouth disease is extremely contagious, and entire herds containing infected animals are destroyed when an outbreak of the disease is confirmed. The economic consequences of a foot-and-mouth disease epidemic can thus be quite severe. Vaccines have been produced which confer a measure of protection against infection by FMDV, but the disease persists in many areas.
The picornaviruses follow a generally similar pattern of replication. First, infectious virus particles bind non-covalently to specific receptors on the surface of a target cell. Virus particles then penetrate the cell membrane and uncoat a single-stranded viral RNA molecule. This positive-stranded RNA initially serves as an mRNA template for synthesis of viral proteins, including a viral RNA polymerase. The viral RNA polymerase catalyzes synthesis of minus-stranded, or complementary, RNA's, which serve as templates for subsequent production of additional plus strands. As the process of infection proceeds, proportionately more of the newly-synthesized plus strands are incorporated into mature virions.
In addition to a viral RNA polymerase, the viral genome also specifies four major capsid structural proteins, designated VP.sub.1, VP.sub.2, VP.sub.3 and VP.sub.4 ; an RNA capping protein designated VP.sub.G ; and several non-structural proteins, including a virus-specified protease. The virus-specified protease plays a unique role in picornavirus replication, and provides a target for design of antiviral compounds which inhibit virus-specified protease activity.
Following a virus-induced shutdown of host cell protein synthesis, viral mRNA is translated to viral protein in a continuous passage of host ribosomes along viral mRNA templates. The resulting translation product is a polyprotein containing several domains, each having a different function. This polyprotein is cleaved, prior to dissociation of the ribosome/protein complex, by host cell and virus-specified proteases. Nascent cleavage reactions, which occur essentially instantaneously, are apparently mediated by cellular proteases associated with the host cell translation apparatus. A series of intermediate cleavage reactions, which induce conformational changes and other alterations in tertiary structure eventually culminating in capsid assembly, are then catalyzed by a highly specific, virus-coded protease. Proteolytic cleavages also regulate RNA synthesis by activating and deactivating the viral RNA polymerase.
The existence of a unique, virus-specified protease in cells infected by picornavirus has been demonstrated by several lines of inquiry. First, as detailed below, the virus-specified protease activity is highly site-specific, and does not resemble the proteolytic activity associated with normal cellular degradative pathways. Second, this site-specific enzyme activity is not detected in extracts of uninfected cells, but is found in lysates of infected cells in quantities which increase both as the process of infection proceeds, and as the quantity of infecting virus is increased. Third, cell-free protein synthesizing systems, programmed with viral mRNA, produce a characteristic protease activity which also processes capsid polypeptides. Fourth, it has been demonstrated in both infected cells and in cell-free systems that the viral protease is not efficient at cleaving proteins of heterologous viruses or cellular proteins.
As noted above, the virus-specified protease is highly site-specific. In the past several years, the amino acid sequences of cleavage sites recognized by virus-specified proteases have been determined for several picornaviruses. This data has been in part provided by end-group analyses of viral proteins, but additional information has been provided by sequencing viral genomic RNA, or DNA complementary to viral RNA. Comparisons of cleaved sites in picornaviruses have revealed several classes of cleavage recognition sites. The nascent cleavage sites processed essentially instantaneously during translation resemble chymotrypsin cleavage sites; new carboxyl termini are typically donated by aromatic or hydrophobic residues. Intermediate, or subsequently-processed cleavage sites, which are recognized by a virus-specified protease, are quite distinct. In the case of picornaviruses, these sites are characterized by a glutamine-glycine or glutamic acid-X linkage, frequently surrounded by hydrophobic (leucine, isoleucine, or valine) sequences. A high degree of conservation among the cleavage site sequences recognized by virus-specified protease suggests that artificial substrates, designed to mimic the structure of the conserved sequence, can be employed to inhibit specific protease activity, and hence, disrupt the process of picornavirus replication.
A number of workers have sought specific inhibitors of picornavirus protease activity. Korant, J. Virol. 10: 751-759 (1972), discloses inhibition of poliovirus and echovirus-12 protein processing by chloromethyl ketone derivatives of simple amino acids. Specifically, Korant discloses inhibition by tolylsulfonylphenylalanyl chloromethyl ketone (TPCK) and tolylsulfonyllysyl chloromethyl ketone (TLCK). Summers et al., J. Virol. 10: 880-884 (1972), similarly disclose inhibition of protease cleavage of large poliovirus-specific polypeptides by TPCK, TLCK, and D- and L-isomers of carbobenzyloxyphenylalanyl chloromethyl ketone (ZPCK). In a subsequent report, Korant et al., Proc. Natl. Acad. Sci. USA 76:2992-2995 (1979), describe inhibition of poliovirus protein processing by carbobenzyloxyleucyl chloromethyl ketone (ZLCK).
Togaviruses are a family of RNA-containing viruses, typically arthropod-borne, which include the pathogens of yellow fever, rubella (measles), dengue fever, encephalitis, and certain subclinical conditions. Like picornaviruses, togaviruses employ protein cleavages to form all viral polypeptides, and evidence exists that a specific protease is coded by the togavirus genome. As in the case of picornaviruses, highly conserved peptide regions are observed at cleavage sites of viral precursor polypeptides, suggesting a role for a virus-specified protease.
RNA-containing tumor viruses, or retroviruses, include a number of genera linked to various sarcomas, leukemias, lymphomas and other carcinomas in avian and mammalian species. Adenoviruses are DNA-containing viruses which induce latent infections in lymphoid tissues, which occasionally erupt into acute episodes of respiratory and ocular infection. Like picornaviruses and togaviruses, retroviruses and adenoviruses appear to encode a specific viral protease.
Various peptide derivatives with capacity to inhibit protease activity are known. Powers, "Haloketone Inhibitors of Proteolytic Enzymes", in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, Weinstein, ed., (Marcel Dekker, New York, 1977), pp. 65-178, has surveyed the literature relating to inhibition of protease activity by haloketone derivatives of amino acids and peptides. Powers et al., Biochim. Biophys, Acta 480: 246-261 (1977), disclose inhibition of subtilisin BPN', a bacterial protease, by a series of peptide chloromethyl ketones. Of the compounds tested, acetyl-L-phenylalanyl-L-glycyl-L-alanyl-L-leucyl cholormethyl ketone (Ac-Phe-Gly-Ala-LeuCH.sub.2 Cl) was the fastest inhibitor. A related compound, methoxysuccinyl-L-phenylalanyl-L-glycyl-L-alanyl-L-leucyl chloromethyl ketone (MeOSuc-Phe-Gly-Ala-LeuCH.sub.2 Cl), is disclosed by Enzyme Systems Products in a November 1981 product bulletin.
Ito et al., Biochem. Biophys, Res. Commun. 49: 343-349 (1972), describe experiments involving inhibition of chymotrypsin, a digestive protease, by certain peptide aldehydes. Ito et al. also tested for inhibition of chymotrypsin by Ac-Leu-Leu-PheCH.sub.3, a tripeptidyl methyl ketone. However, no inhibition was observed at an inhibitor concentration of 600 .mu.g/mL.
Finally, Fittkau et al., "Synthesis and Properties of Peptide Ketones", in Peptides 1982, Blaha et al., eds., (de Gruyter, New York, 1983) pp. 617-622, disclose inhibition of thermitase, a thermostable serine protease of Thermoactinomyces vulgaris, by certain peptide methyl ketones.
A general method for preparing inhibitors of virus-specified proteases has now been established. This method represents a new approach to treatment of viral infection in animals.